The objective of this proposal is to understand the cellular and molecular mechanisms that control a fundamental event in neural development: the switch from neurogenesis to gliogenesis. An understanding of this process is essential for applying neural stern and progenitor cell biology to the treatment of neurological disease. This switch will be studied in a specific population of spinal cord precursors that sequentially generate motoneurons (MNs) and oligodendracytes (oligos). These precursors can be prospectively isolated using fluorescence-activated cell sorting (FACS), by means of a GFP reporter expressed from the Olig2 locus, which encodes a transcription factor required for both MN and oligo differentiation. Using these isolated cells, we will address the following specific aims: I). We will test whether MNs and oligos develop from a multipotential, self-renewing stem cell in the ventricular zone (VZ) of the spinal cord, as is widely assumed, or rather from progenitors that undergo irreversible restrictions in developmental competence. We will investigate this by using a newly developed technique for direct transplantation of freshly isolated Olig2-expressing progenitors into the chick spinal cord, without any ex vivo expansion (which may perturb the properties of the cells). Using this approach, we will perform heterochronic transplantation experiments to test the self-renewal anc developmental capacities of Olig2-i- cells at different stages during the MNgoligo transition. II). We will test the hypothesis that changes in gene expression in Olig2+ progenitors during the neuron-to-glial switch reflect the regulation of several distinct subclasses of genes, each with different kinetics of activation and repression. This hypothesis will be tested by using oligonucleotide microarrays to perform gene expression profiling (GEP) experiments on acutely isolated Olig2+ progenitors from different stages of spinal cord development. This GEP analysis should also identify a) markers useful in clarifying lineage relationships between Olig2+ progenitors of MNs and oligos;and b) candidate regulatory genes for functional analysis. Ill) We will tost the hypothesis that there is a "temporal code" of transcription factors that controls the MN->oligo switch, by performing gain-of-function (GOF) and loss-of-function (LOF) genetic manipulations of candidate regulatory genes identified in the GEP temporal analysis (Aim II). Electroporation of chick spinal cord will be used as a rapid in vivo assay for such functional manipulations, employing expression of full-length cDNAs, and independently validated shRNAi (small hairpin RNAi) constructs, for GOF and LOF experiments, respectively. The embryos will be analyzed uning an extensive battery of molecular markers for various classes of neurons (including MNs and interneurons), oligodendrocytes, and newly validated markers of astrocytes and their progenitors. Candidates for which strong functional data is obtained from the chick system will be further validated by generating constitutive or conditional knockouts in the mouse. IV) We will test the hypothesis that targets of OLIG2, which functions as a transcriptional repressor, include a) repressers of oligo differentiation;and b) activators of astrocyte differentiation. Candidate targets of OLIG2 will be identified by performing comparative GEP analysis of isolated Olig2-GFP+ cells from Olig1/2+/- and Olig2-/- spinal cord, at several developmental stages. A series of analytic algorithms will be used to filter the data to obtain a list of transcription factors that are de-repressed in the absence of OLIG2 function. These candidates will be further validated and prioritized by real-time RT-PCR and in situ hybridization. Top candidates will then be functionally analyzed by GOF and LOF manipulations in chick embryos.